Single-Cell Probing of Nanoscale Bacterial Adhesion in Real-Time Using Optical Tweezers

Bacterial biofilms are organized microbial communities that profoundly impact medicine, industry, and microbial ecology. Biofilm formation begins with nanoscale adhesion events between single bacteria and a surface, and the earliest stages of surface colonization involve reversible interactions that transition to irreversible attachment through the secretion of specialized nanoscale bioadhesins. Understanding and controlling the initial interactions between adhesin and surface is key to control and prevent biofilm formation. Here, we investigate the nanoscale adhesion dynamics of single Caulobacter crescentus cells, a dominant early colonizer in environmental biofouling, focusing on its holdfast, a strong nanoscale adhesive organelle that mediates irreversible attachment within seconds of contact. To characterize the time-dependent mechanical properties of holdfast, we developed the Trapezoid, a custom optical tweezers platform that combines nanometer spatial precision with millisecond temporal control of cell position, contact timing, and applied force on defined surfaces. We implemented a trapezoidal temporal profile of these programmed contact cycles, in which a single cell is brought into contact with the surface, maintained for a defined duration, retracted, and subjected to controlled pulling forces. This approach enables real-time quantification of nanoscale adhesion onset, holdfast deployment kinetics, and influence of surface chemistry at the level of individual live adhesion events. Our results dissect the physical and biochemical determinants of bacterial adhesion, providing a quantitative framework for the rational design of antiadhesive coatings, nanoscale biofouling control strategies, and bioinspired adhesives functional in wet environments.